The long-term goal of this project is to understand at cellular and molecular levels the pathogenesis of neuroblastoma, a common childhood malignant tumor of neural crest origin that arises in the sympathetic nervous system. For this grant period, we will focus on Bmi-1, which is essential for the maintenance of stem cells through transcriptional repression of genes that control stem cell self-renewal proliferation, differentiation, apoptosis, and senescence. Based on the data from our preliminary studies, we hypothesize that Bmi-1 contributes to neuroblastoma development by maintaining sympathetic neural crest stem cells, from which neuroblastoma probably derives, and neuroblastoma stem cells, which drive tumor growth. We will test the hypothesis in both animal- and cell-based systems. In studies under AIM 1, we will define the role of Bmi-1 in neuroblastoma development. We will examine the effects Bmi-1 deficiency on neuroblastoma initiation and progression in MYCN mice, an animal model of the human disease. We will further assess its role in development of the stromal microenvironment suitable for neuroblastoma genesis. In studies under AIM 2, we will define the cellular basis for the oncogenic activity of Bmi-1 in neuroblastoma initiation and progression. We will examine the effects of Bmi-1 deficiency on malignant transformation and maintenance of sympathetic neural crest stem cells and neuroblastoma stem cells. We will determine how Bmi-1 deficiency may affect stem cell fate decisions: proliferation, differentiation, apoptosis, or senescence. In studies under AIM 3, we will elucidate the molecular mechanisms for the oncogenic activity of Bmi-1 in the pathogenesis of neuroblastoma. We will examine the role of Ink4a and ARF as tumor suppressors in neuroblastoma development in MYCN mice and as Bmi-1 targets in regulation of neuroblastoma cell proliferation and senescence. We will examine the role of Bim as a Bmi-1 target in regulation of neuroblastoma cell apoptosis. Finally, we will examine the role of HoxC9 as a Bmi-1 target in regulation of neuroblastoma cell differentiation. These studies should further our understanding of the molecular basis underlying neuroblastoma genesis and may suggest new targets for development of more effective and specific therapeutic agents for this deadly childhood cancer.